The present invention relates to recombinant DNA that encodes the BseRI restriction endonuclease (endonuclease) as well as BseRI methyltransferase (methylase), expression of BseRI endonuclease and methylase in E. coli cells containing the recombinant DNA.
BseRI endonuclease is found in the strain of Bacillus species R (CAMB2669) (New England Biolabs"" strain collection). It recognizes the double-stranded DNA sequence 5xe2x80x2GAGGAG3xe2x80x2 N10/N8 (SEQ ID NO:1) and cleaves the downstream sequence at N10 of the top strand and N8 of the bottom strand, generating a 2-base 3xe2x80x2 overhang (N=A, C, G, or T). BseRI methylase (M.BseRI) is also found in the same strain. It recognizes the double-stranded DNA sequence 5xe2x80x2GAGGAG3xe2x80x2 (SEQ ID NO:1) and presumably modifies the N6 adenine on the top strand and the N4 cytosine on the bottom strand of 5xe2x80x2CTCCTC3xe2x80x2 (SEQ ID NO:2).
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases recognize and bind particular sequences of nucleotides (the xe2x80x98recognition sequencexe2x80x99) along the DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and eleven restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 27:312-313, (1999)).
Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5xe2x80x2TTT/AAA3xe2x80x2 (SEQ ID NO:3), 5xe2x80x2PuG/GNCCPy3xe2x80x2 (SEQ ID NO:4) and 5xe2x80x2CACNNN/GTG3xe2x80x2 (SEQ ID NO:5) respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5xe2x80x2G/AATTC3xe2x80x2 (SEQ ID NO:6).
A second component of bacterial/viral restriction-modification (R-M) systems are the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiably foreign DNA, is sensitive to restriction endonuclease recognition and cleavage. During and after DNA replication, usually the hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction digestion.
With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, i.e. populations of clones derived by xe2x80x98shotgunxe2x80x99 procedures, when they occur at frequencies as low as 10xe2x88x923 to 10xe2x88x924. Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719, (1980); HhaII: Mann et al., Gene 3:97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507, (1981)). Since the expression of restriction-modification systems in bacteria enables them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509, (1985); Tsp45I: Wayne et al. Gene 202:83-88, (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421, (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225, (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119, (1983); and MspI: Walder et al., J. Biol. Chem. 258: 1235-1241, (1983)).
A more recent method, the xe2x80x9cendo-blue methodxe2x80x9d, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (U.S. Pat. No. 5,498,535 (1996); Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). This method utilizes the E. coli SOS response signals following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535). The disadvantage of this method is that some positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.
There are three major groups of DNA methyltransferases based on the position and the base that is modified (C5 cytosine methylases, N4 cytosine methylases, and N6 adenine methylases). N4 cytosine and N6 adenine methylases are amino-methyltransferases (Malone et al. J. Mol. Biol. 253:618-632, (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer the DNA site resistant to restriction digestion. For example, Dcm methylase modification of 5xe2x80x2CCWGG3xe2x80x2 (SEQ ID NO:7) (W=A or T) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpM methylase can modify the CG dinucloetide and make the NotI site (5xe2x80x2GCGGCCGC3xe2x80x2 (SEQ ID NO:8)) refractory to NotI digestion (New England Biolabs"" Catalog, 2000-01, page 220). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.
Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant molecules in the laboratory, there is a strong commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes. Such over-expression strains should also simplify the task of enzyme purification.
The present invention relates to a method for cloning the BseRI restriction endonuclease from Bacillus species R into E. coli by direct PCR and inverse PCR amplification from genomic DNA.
It proved difficult to clone bseRIM gene by the conventional methylase selection method. At first, Sau3AI partial genomic DNA library, AatII, BamHI, and PstI complete genomic DNA libraries were constructed. After BseRI challenge, no true methylase positive clones were identified among the surviving transformants. Since the conventional methylase selection did not yield any positive clones, efforts were made to purify the native BseRI endonuclease.
BseRI endonuclease was purified from the native strain Bacillus cell extract by chromatography through Heparin hyper D, Source Q, Heparin tsk columns and gel filtration column Superdex 75. Two major proteins were identified on SDS-PAGE, one at xcx9c55 kDa and the other at xcx9c120 kDa. Both proteins were subjected to protein sequencing to obtain the N-terminus amino acid sequence. Amino acid sequence comparison with proteins in GenBank indicated that the xcx9c55 kDa protein has high homology to Basillus Glutaminyl tRNA sythetase. Therefore, this protein was ruled out as the BseRI endonuclease. The N-terminal amino acid sequence of the xcx9c120 kDa protein was sequenced and the sequence has no significant homology to proteins in GenBank. It was concluded that the xcx9c120 kDa protein is most likely the BseRI endonuclease.
A protein at xcx9c46 kDa was also identified in the production preparations of BseRI endonuclease (lot 8, 9, and 12). This protein was also sequenced, which generated a similar N-terminus amino acid sequence to the xcx9c120 kDa. The xcx9c46 kDa protein might be a protease degraded fragment of the xcx9c120 kDa protein. Degenerate primers were synthesized based on the amino acid sequence. The 92-bp coding DNA was amplified by PCR using degenerate primers and cloned into a pUC-derivative and sequenced. The predicted amino acid sequence from the DNA sequence matched very well the actual amino acid sequence derived from the BseRI protein.
Inverse PCR and DNA sequencing were performed to obtain the remaining part of the bseRIR gene. After five round of inverse PCR amplifications and DNA sequencing the entire bseRIR endonuclease gene was sequenced and found to be 3345 bp, encoding a fusion protein with a restriction domain, a conserved methylase domain, and a specificity domain (R-M-S).
Because R-M genes in a particular R-M system are usually located in close proximity, efforts were made to identify the adjacent DNA sequences. After four rounds of inverse PCR amplifications, a large ORF of 3930 bp was found upstream of bseRIR gene. This large ORF encodes two amino-methylases (a N4 cytosine methylase and a N6 adenine methylase) that fused together to form BseRI methylase.
A pre-modified expression host ER2566 [pACYC-BseRIM] was constructed. The bseRIR gene was amplified by PCR from genomic DNA and cloned into a T7 expression vector pAII17. The expression strain was ER2566 [pACYC-BseRIM, pAII17-BseRIR]. An induced BseRI endonuclease protein band of approximately 120-125 kDa was detected in the IPTG-induced cell extract, but absent in the non-induced extract. The cell extract was confirmed to display recombinant BseRI endonuclease activity on xcexDNA.